S120: MNX1-ACTIVATING ENHANCER HIJACKING EVENTS IN ACUTE MYELOID LEUKEMIA WITH DELETIONS ON CHROMOSOME 7Q
Authors: Anna Riedel, Justyna A. Wierzbińska, Umut H. Toprak, Aurore Touzart, Etienne Sollier, Simge Kelekçi, Katherine Kelly, Dieter Weichenhan, Anastasija Pejkovska, Ashish Goyal, Charlotte Meinen, Matthias Schlesner, Frank Westermann, Benedikt Brors, Lars Bullinger, Philipp Greif, Michael Lübbert, Florian Heidel, Thomas Fischer, Claudia Gebhard, Brigitte Schlegelberger, Gudrun Göhring, Yvonne Behrens, Ekaterina Jahn, Hartmut Döhner, Konstanze Döhner, Ruud Delwel, Pavlo Lutsik, Daniel B. Lipka, Christoph Plass
Journal: HemaSphere (2023)

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Deletions in the long arm of chromosome 7 (del7q) are recurrent events in acute myeloid leukemia (AML) which are associated with an unfavorable outcome. Since the search for recessive tumor suppressor genes located within the deleted region was unsuccessful, the prevalent idea currently is that haploinsufficiency of one or more tumor suppressor genes drive this leukemia. Other studies showed that structural rearrangements such as translocations and inversions can lead to the activation of oncogenes through relocation of cis-regulatory elements. A similar mechanism would also be plausible for chromosomal deletions, specifically del(7q) in AML. Aims: We investigated the hypothesis that del(7q) could activate a proto-oncogene located outside the deleted region due to restructuring of topologically associating domains and relocation of cis-regulatory elements. Methods: Whole genome sequencing (WGS), RNA sequencing (RNAseq) and Infinium MethylationEPIC array were used to study 13 patients with isolated del(7q) or monosomy 7. Epigenomic profiling using antibody-guided chromatin tagmentation with sequencing (ACT-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq) and circular chromosome conformation capture (4C) was done on primary AML samples with MNX1 activation. CRISPR/Cas9 genome editing was used to generate model systems for the putative enhancer hijacking event. Results: We identified a subgroup of del(7q)-AML that aberrantly expresses Motor Neuron and Pancreas Homeobox (MNX1), which is a key developmental homeobox gene located on chromosome 7q36.3 coding for the transcription factor MNX1. MNX1 expression is usually tightly regulated and restricted to the brain, gastrointestinal tract and pancreas. The majority of MNX1-expressing cases had breakpoints within CDK6 and upstream of MNX1, thus, the deletions juxtapose MNX1 with a region containing a strong hematopoietic enhancer. To verify the presence of an enhancer near the breakpoint, we used epigenomic techniques to map accessible chromatin and histone modifications in primary AML. We identified a putative enhancer in CDK6 and demonstrated interaction between the CDK6 region and the MNX1 promoter in these samples. To strengthen the evidence that the enhancer drives MNX1 activation, we inserted the putative enhancer upstream of MNX1 in a human induced pluripotent cell line (hiPSC). Upon differentiation into CD34+ hematopoietic stem and progenitor cells (HSPC), MNX1 was activated in the hiPSC line containing the inserted enhancer. This activation was not observed in the control, i.e. the parental hiPSC line without the inserted enhancer. Summary/Conclusion: We showed that chromosomal deletions in AML can lead to enhancer hijacking events through reshuffling of cis-regulatory elements. Specifically, we showed that MNX1 is activated in a subgroup of del(7q)-AML by CDK6 enhancer. Further validation and insights into the effects of MNX1 activation will be gained from single cells RNA sequencing (scRNA-seq) and single cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) in the hiPSC line with the inserted enhancer fragment. Keywords: Gene regulation, Cytogenetic abnormalities, Acute myeloid leukemia, Epigenetic